Toxic enzymes from plants and bacteria such as ricin, diphtheria toxin and Pseudomonas toxin have been coupled to antibodies or receptor binding ligands to generate cell-type-specific-killing reagents (Youle, et al., Proc. Nat""l Acad. Sci. USA 77:5483 (1980); Gilliland, et al., Proc. Nat""l Acad. Sci. USA 77:4539 (1980); Krolick, et al., Proc. Nat""l Acad. Sci. USA 77:5419 (1980)). Regardless of the fact that the cell-recognition moiety is not always an antibody, these directed toxins are generally known as immunotoxins. These hybrid proteins kill cells which express the receptor or cell surface marker that the antibody or ligand portion of the molecule recognizes.
Under appropriate conditions, depending on the particular receptor or cell marker, the toxin enters the cytosol, inactivates the protein synthesis machinery and causes death of the target cell. Immunotoxins, which have been shown to be highly cytotoxic to cancer cells growing in cell culture and in animal models, demonstrate the potential of these reagents to treat blood and lymph borne malignancies which, because of their dissemination are not treatable by traditional surgical techniques, as well as solid tumors in restricted compartments such as the intraperitoneal cavity (reviewed in Griffin, et al., IMMUNOTOXINS, p 433, Boston/Dordrecht/Lancaster, Kluwer Academic Publishers, (1988); Vitetta, et al., Science 238:1098 (1987); Fitzgerald, et al., J. Nat""l Cancer Inst. 81:1455 (1989)). Traditional chemotherapies, while being effective in the treatment of some cancerous conditions, exhibit undesired side effects due to the systemic toxicity of the chemotherapeutic compounds.
An ideal candidate for cancer therapy, therefore, would be an immunotoxin that would selectively be cytotoxic to cancer cells yet remain harmless to non-cancerous cells of the patient. Utilization of this type of anti-tumor therapy, however, has been stymied by the development of immune responses in patients to foreign proteins which comprise the immunotoxins. Immune responses against murine monoclonal antibodies (Sawler, et al., J. Immunol. 135:1530 (1985); Schroff, et al., Cancer Res. 45:879 (1985)) and anti-toxin antibodies have been detected in both animals and humans treated with immunotoxins (Rybak, et al., Immunol. and Allergy Clinics of North America 11 (2):359 (1991); Harkonen, et al., Cancer Res. 47:1377 (1987); Hertler, A. in IMMUNOTOXINS p. 475, Kluwer Academic Publishers, Boston/Dordrecht/Lancaster (1988)). Advances in humanization techniques have alleviated some of the immunogenicity associated with the antibody portion of immunotoxins (Bird, et al., Science 242:423 (1988); Huston, et al, Proc. Nat""l Acad. Sci. USA 85:5879 (1988); Ward, et al., Nature 341:544 (1989); and Jones, et al., Nature 314:522 (1986)). However, no solution has been found to counter the immunogenicity of the toxic moiety other than immunosuppression of the patients (Khazaeli, et al., Proceedings of AACR 29:418 (1988)). Thus, there has been a continuing need for methods and compositions that would reduce the immunogenicity of the toxic moiety of immunotoxins yet retain the ability to selectively kill cells having a given surface marker.
Non-Hodgkin""s lymphomas fall mostly under the generic rubric of B-cell lymphomas and can either be a disseminated or a solid tumor within the lymph system. Radiolabeled humanized murine antibodies which have been raised against CD22 (LymphoCide(trademark)), a lineage-restricted surface marker on malignant and normal B cells, are currently in clinical trials as a treatment for B-cell lymphomas and certain autoimmune diseases which can be affected by selecting depleting the normal B-cell population that produces the autoantibodies involved in the pathogenesis of these autoimmune diseases, such as systemic lupus erythematosis and Sjxc3x6gren""s syndrome. See also, Amlot, et al., Blood 82:2624-2633 (1993); Sausville, et al., Blood 85:3457-3465 (1995); Grossbard, et al., Blood 81:2263-2271 (1993); Grossbard, et al., Clin. Oncol. 11:726-737 (1993). To date, some antitumor responses have been noted but immunotoxin-mediated toxicity to normal tissue often prevented dosing at therapeutic levels. In addition to CD22, several other B-cell-specific antigens such as CD19 and CD40 have been targeted by immunotoxins made with plant toxins such as ricin A-chain and bacterial toxins, such as Pseudomonas exotoxin A (PE). Uckun, et al., Blood 79:2201-2214 (1992); Ghetie, et al., Cancer Res. 51:5876-5880 (1991); Francisco, et al, Cancer Res. 55:3099-3104 (1995).
The cytotoxicity of RNase A toward tumor cells is well documented from studies performed in the 1960s and 1970s. Early work is reviewed in Roth, Cancer Res. 23:657 (1963). The relevance of these early studies has been sustained by the discovery that an anti-tumor protein from oocytes of Rana pipiens is homologous to bovine pancreatic RNase A (Ardelt, et al., J. Biol Chem, 256:245 (1991)). P- 30 protein (and referred to herein as the onc protein) was isolated from extracts of Rana pipiens early embryos based upon anti-proliferative/cytotoxic effects toward cancer cells in vitro (Darzynkiewicz, et al., Cell Tissue Kinet. 21:169 (1988); Mikulski, et al., Cell Tissue Kinet. 23:237 (1990)) and in animal models (Mikulski, et al., J. Nat""l Cancer Inst. 82:151 (1990)). Phase III human clinical trials of the onc protein in patients with a variety of solid tumors are currently in progress.
In one embodiment, the present invention relates to cytotoxic reagents comprising an antibody and a moiety having ribonucleolytic activity derived from a non-human ribonuclease. The inventor has found that these particular immunotoxins had highly surprising properties as they were up to 2000 fold more active against malignant B cells than their human RNase counterparts or than the toxin itself. Further, as will be described in more detail below, their use when administered in vivo against disseminated tumors, resulted in dramatically lowered side effects. These highly effective, but apparently non-toxic, immunotoxins directed against such ubiquitous diseases as B cell lymphomas present a new and exciting therapeutic option for patients suffering from such diseases.
Another embodiment is a cytotoxic reagent comprising an internalizing antibody and a moiety having ribonucleolytic activity, wherein the internalizing antibody binds to a lineage-dependent antigen or an antigen associated to a greater extent with cancer cells than with normal cells.
A further object of the present invention to provide cytotoxic ribonuclease (RNAse) immunotoxins that selectively kill cells having a given surface marker. These immunotoxins are minimally immunogenic and generate less systemic toxicity than presently known immunotoxins. In particular, it is an object of the present invention to provide direct immunotoxins comprising protein fragments with ribonucleolytic activity linked to humanized antibodies that recognize specific markers on or in tumor cells.
In another embodiment, the present invention relates to a pharmaceutical composition comprising an immunotoxin of the present invention and a pharmaceutically acceptable carrier.
In a further embodiment, the present invention relates to a method of selectively killing cancer cells. The method comprises contacting the tumor cells to be killed with a selective immunotoxin of the present invention under conditions such that a monoclonal antibody binds to a surface or intracellular marker on or in the tumor cell, thereby causing the toxic ribonuclease to kill the cell.
In a still further embodiment, the present invention relates to a method of selectively killing normal cells involved in pathological processes. The method comprises contacting the normal cells to be killed with a selective immunotoxin of the present invention under conditions such that a monoclonal antibody binds to a surface or intracellular marker in or on the normal cell, thereby causing the toxic ribonuclease to kill the cell.
Various other objects and advantages of the present invention will be apparent from the following description of the invention.